The invention relates to a dye-labeled oligonucleotide for labeling a nucleic acid molecule having a target sequence section, the dye-labeled oligonucleotide having the following components: a loop section which has a loop sequence essentially complementary to said target sequence; a first stem section having at least three nucleosides and arranged at one end of the loop section; a second stem section having at least three nucleosides and arranged at the other end of the loop section, the two stem sections being able to hybridize intramolecularly; and a fluorophore which is bound to a position of the first stem section.
The invention further relates to the use of the dye-labeled oligonucleotide for labeling in a solution a nucleic acid molecule having a target sequence section and for two methods for detecting in a solution a nucleic acid molecule having a target sequence section.
The dye-labeled oligonucleotides mentioned at the outset are frequently called “nucleic acid probes”. They play a central part in the rapid and sensitive detection of specific known nucleic acid molecules (DNA or RNA) in biological samples in molecular biology and biotechnology. Specific applications include, inter alia, medical early recognition of a bacterial or viral infection, forensics, use in DNA/RNA amplification by PCR or by other techniques, in the early diagnosis of a genetic defect and in discriminating between similar organisms and alleles.
A variety of methods for detecting nucleic acids and determining the amount of nucleic acids are known. The ubiquitous Southern blotting method is characterized by time-consuming steps and poor sensitivity.
A relatively new elegant method for detecting a specific nucleic acid molecule uses “molecular beacons” (Tyagi et al. 1996, Nature Biotechnology 14, 303–308; Kostrikis et al. 1998, Science 279, 1228–1229). Molecular beacons are dye-labeled oligonucleotides which have the stem-loop structure mentioned at the beginning. A fluorophore is coupled to each of the two free ends of the stem sections (3′ and 5′ ends). One fluorophore serves as fluorescent dye and the other one as quenching dye, which quenches the fluorescence of the fluorescent dye via Foerster energy transfer when in sufficiently close spatial proximity.
The sequences of the stem sections on both ends of the molecular beacons are chosen such that, when the molecular beacon folds, the stem sections hybridize exclusively with one another but not with other sections of the oligonucleotide. In the state of hybridized stem sections, the distance between the fluorescent dye and the quenching dye is sufficiently short so that the fluorescent dye does not fluoresce, even with suitable excitation with light.
The loop section has a sequence, which is complementary to the sequence of the target sequence section. If the molecular beacons and the DNA/RNA molecules having the target sequence are in a solution together, it is possible for the loop sections and the target sequence sections to hybridize. The sequences and lengths of the stem sections and loop sections are chosen such that the molecular beacon unfolds with breaking-up of the hybridization of the two stem sections. Due to said unfolding, the spatial distance between the fluorescent dye and the quenching dye is greatly increased. The fluorescent dye can then be excited to emit fluorescence.
If the fluorescence intensity of the fluorescent dye is observed continuously, an increase can be detected when the molecular beacons detect the target sequence sections of the nucleic acid molecules and hybridize therewith. In this way it is possible to detect the nucleic acid molecules quantitatively.
The disadvantage of this type of molecular beacons is their relatively complicated synthesis, since the oligonucleotide must be labeled specifically with the fluorescent and the quenching dye both on the 5′ and on the 3′ end.
It would therefore be desirable and advantageous to provide an improved dye-labeled oligonucleotide to obviate prior art shortcomings and to provide a suitable dye-labeled oligonucleotide for detecting nucleic acids in solution.